N. meningitidis is an important cause of meningitis and sepsis. Conventional approaches to develop a vaccine for prevention of disease caused by capsular group B strains, which account for 30-80% of all cases, have been largely unsuccessful. We propose to investigate two complementary vaccine approaches. First is a subunit vaccine based on our studies of three recombinant proteins, Neisserial protein A (NspA) and two antigens, designated NspD and E, which were discovered during the group B MC58 genome sequencing project. All three elicit bactericidal antibody responses against homologous and heterologous strains and, therefore could form the basis of monovalent or multivalent vaccines. Our second approach is based upon our discovery that sequential immunization with native vesicles prepared from three meningococcal strains, each differing by capsular group, PorA variable region sequence type, PorB serotype and LOS immunotype, overcomes strain-specific bactericidal antibody responses associated with conventional prime/boost immunization with vesicles vaccines, in part by enhancing the antibody responses to NspA. The anti-NspA antibody responses to sequential immunization with vesicle vaccines also are higher than those made to recombinant NspA. Our hypothesis is that repeated presentation of vesicle vaccines containing highly conserved antigens in the context of different, variable antigens such as PorA, which is usually immunodominant, enhances the response to the conserved antigen. In Aim 1, we will investigate improving the ability of recombinant NspA, D, or E to elicit protective antibody responses by reconstituting the recombinant proteins in liposomes or micelles to restore conformational epitopes. We also will prepare a panel of bactericidal mAbs to monitor expression of critical epitopes in the recombinant proteins, or improved vesicle vaccines (Aim 2). In Aim 2, we will construct specialized N. meningitidis strains to produce improved vesicle vaccines that are designed to both enhance immunogenicity by over-expressing NspA, D and E, and to minimize toxicity by genetically decreasing unwanted or potentially toxic antigens. In Aim 3, we will determine whether vaccines composed of more than one recombinant antigen, or vesicle vaccines combined with a recombinant antigen, can augment bactericidal antibody responses. The proposed studies will identify safe and more broadly protective vaccine strategies for prevention of N. meningitidis disease, including group B strains for which there is currently no vaccine available. The lessons learned also will be broadly applicable to advancing vaccines against other pathogens that target antigens identified by "genome mining." [unreadable] [unreadable]